1. Field of the Invention
The present invention relates to a purified thermostable enzyme. In one embodiment the enzyme is DNA polymerase purified from Thermus aquaticus and has a molecular weight of about 86,000-95,000. In another embodiment the enzyme is DNA polymerase produced by recombinant means.
2. Background Art
Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al., J. Biol. Chem. (1957) 233:171-177 and Buttin and Kornberg (1966) J. Biol. Chem. 241:5419-5427.
In contrast, relatively little investigation has been made on the isolation and purification of DNA polymerases from thermophiles, such as Thermus aquaticus. Kaledin et al., Biokhymiya (1980) 45:644-651 discloses a six-step isolation and purification procedure of DNA polymerase from cells of T. aquaticus YT1 strain. These steps involve isolation of crude extract, DEAE-cellulose chromatography, fractionation on hydroxyapatite, fractionation on DEAE-cellulose, and chromatography on single-strand DNA-cellulose. The pools from each stage were not screened for contaminating endoand exonuclease(s). The molecular weight of the purified enzyme is reported as 62,000 daltons per monomeric unit.
A second purification scheme for a polymerase from T. aquaticus is described by A. Chien et al., J. Bacteriol. (1976) 127:1550-1557. In this process, the crude extract is applied to a DEAE-Sephadex column. The dialyzed pooled fractions are then subjected to treatment on a phosphocellulose column. The pooled fractions are dialyzed and bovine serum albumin (BSA) is added to prevent loss of polymerase activity. The resulting mixture is loaded on a DNA-cellulose column. The pooled material from the column is dialyzed and analyzed by gel filtration to have a molecular weight of about 63,000 daltons, and, by sucrose gradient centrifugation of about 68,000 daltons.
The use of a thermostable enzyme to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present has been suggested in U.S. Pat. No. 4,683,195. Primers, nucleotide triphosphates, and a polymerase are used in the process, which involves denaturation, synthesis of template strands and hybridization. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. The patent discloses thatif the polymerase employed is a thermostable enzyme, it need not be added after every denaturation step, because the heat will not destroy its activity. No other advantages or details are provided on the use of a purified thermostable DNA polymerase. Furthermore, New England Biolabs had marketed a polymerase from T. aquaticus, but was unaware that the polymerase activity decreased substantially with time in a storage buffer not containing non-ionic detergents.
Accordingly, there is a desire in the art to produce a purified, stable thermostable enzyme that may be used to improve the nucleic acid amplification process described above.